We will study two systems at the heart of all biology: RNA Polymerase II and the Ribosome, both studied experimentally by colleagues at Stanford. We are interested in how these machines move as they function and approach the problem by mapping out their conformational state-space using multiscale hybrid methods. Different methods will be needed: in Aim 1, we will assemble these from published studies as well as methods the lab developed and successfully used before. In Aim 2 we will work on RNA pol II comparing results of new methods with trajectories we already have generated. Finally in Aim 3, we will tackle the ribosome. The work is timely in that key functions in the cell are carried out b macromolecular machines. Their structures are being determined rapidly by X-ray crystallography and Cryo-EM: proven tools for analysis and simulation will fully realize the value of this data in advancing biomedicine. Driven by the hypothesis that multi-state multiscale models can simulate functional motion in macromolecular machines, we have 3 specific aims: 1. Validate Hybrid Multiscale Methods for Simulation of Functional Motion. After assembling tools, we will use preliminary studies to advance their capacity to tackle large nucleic-acid protein complexes. Most important will be Molten Zone Molecular Dynamics to avoid periodic boundaries and speed calculations. 2. Determine State-Space of RNA Polymerase II for Translocation in RNA Synthesis. Our existing periodic boundary trajectories and the resulting Markov State Models (MSM) of the space will allow verification of the molten zone molecular dynamics needed for the 5-fold larger ribosome (Aim 3). We will show our methods work by aiding interpretation of experiment and making verifiable predictions. 3. Determine State-Space of The Ribosome for Translocation in Protein Synthesis. We will focus on the prokaryote ribosome. We will morph between states to find relevant transition paths. Following work on RNA pol II, We will sample low energy conformations of every structure and finally we will use Molten Zone molecular dynamics to study the movement from pre- to post translocation states. Given the coming deluge of biological structure data due to recent advances in electron microscopy, our methods are increasingly important in that new structural data will be for large macromolecular complexes rather than the smaller systems conventionally determined by X-ray crystallography. This proposal is significant in that it will facilitate study of the relationship between structure and function in large macromolecular machines, a keystone of modern biomedical science. We believe that existing methods must be combined to tackle a new generation of large and difficult simulation problems. In addition, effort will be focused on new ways to analyze huge amounts of structural data and present it in an easily appreciated manner to a diverse audience of innovative experimental scientists.